Biology subjects

Linser, R.

Publications and source records attributed to Linser, R..

4 recordsLinked to original sources

bioRxiv2023

Transient structural properties of the Rho GDP-dissociation inhibitor

Rho GTPases are master spatial regulators of the cytoskeleton that control a wide range of cellular processes. Their inactivation by removal from cellular membranes involves the stepwise formation of a stable complex with guanine nucleotide dissociation inhibitors (RhoGDIs), for which process the RhoGDI N-terminus is indispensable. The formation of this interface has been thought to emerge from an intrinsically disordered state of RhoGDI in its free, apo form. Here we use tailored solution NMR analyses, molecular dynamics simulations, and biochemical essays to pinpoint the site-specific structural features of full-length RhoGDI1 before and after binding its GTPase client Cdc42. In contrast to the current mechanistic understanding, a diverse set of NMR data unequivocally shows that the structural properties of the GDI N-terminus seen in crystal structures of the complex with GTPases already exist as largely preformed features in free, apo GDI. Even more interestingly, the required structural properties are imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. Lastly, upon Cdc42 binding, the flexibility of the N-terminus and its secondary-structural propensities are not largely abrogated. These observations change the textbook picture of the mechanism of membrane extraction of the GTPase. Rather than a disorder-to-order transition upon binding, an active role of the N-terminus with differentially preformed structural properties, suitably modulated by the specific surrounding along the multi-step process, seems required to leverage the intricate and highly selective extraction process.

biophysics↗

bioRxiv2022

Integrated assessment of structure and dynamics of solid proteins via accurate solid-state NMR distance information

Understanding macromolecular function, interactions and stability hinges on detailed assessment of conformational ensembles. For solid proteins, accurate elucidation of the spatial aspects of dynamics at physiological temperatures are limited by the qualitative character or low abundance of solid-state NMR internuclear distance information. Here, we demonstrate access to abundant proton-proton internuclear distances for integrated structural biology and chemistry with unprecedented accuracy. Apart from highest-resolution single-state structures, the exact distances enable molecular dynamics (MD) ensemble simulations orchestrated by a dense network of experimental inter-proton distance boundaries gathered in the context of their physical lattices. This direct embedding of experimental ensemble distances into MD will provide access to representative, atomic-level spatial details of conformational dynamics in supramolecular assemblies, crystalline and lipid-embedded proteins, and beyond.

biophysics↗

bioRxiv2022

Epigenetic CpG Duplex Marks Probed by an Evolved DNA Reader via a Well-Tempered Conformational Plasticity

5-methylcytosine (mC) and its TET-oxidized derivatives exist in CpG dyads of mammalian DNA and regulate cell fate, but how their individual combinations in the two strands of a CpG act as distinct regulatory signals is poorly understood. Readers that selectively recognize such novel "CpG duplex marks" could be versatile tools for studying their biological functions, but their design represents an unprecedented selectivity challenge. By mutational studies, NMR relaxation, and MD simulations, we here show that the selectivity of the first designer reader for an oxidized CpG duplex mark hinges on precisely tempered conformational plasticity of the scaffold adopted during directed evolution. Our observations reveal the critical aspect of defined motional features in this novel reader for affinity and specificity in the DNA/protein interaction, providing unexpected prospects for further design progress in this novel area of DNA recognition.

genomics↗

bioRxiv2021

Residue-specific insights into (2x)72 kDa tryptophan synthase obtained from fast-MAS 1H-detected solid-state NMR

Solid-state NMR has emerged as a potent technique in structural biology, suitable for the study of fibrillar, micro-crystalline, and membrane proteins. Recent developments in fast-magic-angle-spinning and proton-detected methods have enabled detailed insights into structure and dynamics, but molecular-weight limitations for the asymmetric part of target proteins have remained at ~30-40 kDa. Here we employ solid-state NMR for atom-specific characterization of the 72 kDa (asymmetric unit) microcrystalline protein tryptophan synthase, an important target in pharmacology and biotechnology, chemical-shift assignments of which we obtain via higher-dimensionality, 4D and 5D solid-state NMR experiments. The assignments for the first time provide comprehensive data for assessment of side chain chemical properties involved in the catalytic turnover, and, in conjunction with first-principles calculations, precise determination of thermodynamic and kinetic parameters is demonstrated for the essential acid-base catalytic residue {beta}K87. The insights provided by this study expand by nearly a factor of two the size limitations widely accepted for NMR today, demonstrating the applicability of solid-state NMR to systems that have been thought to be out of reach due to their complexity.

biochemistry↗